Human T-cell lymphotropic virus (HTLV) type I (HTLV-I) was the first retrovirus to be discovered in man in 1980. It is the causative agent of T-cell leukemia and/or lymphoma and of HTLV-associated myelopathy, a severe demyelinating condition that eventually leads to tropical spastic paraparesis. The cumulative lifetime risk of developing these fatal and incurable diseases amounts to ˜5% in asymptomatic carriers of HTLV-I. HTLV-I infects primarily CD4-positive T-cells. It is also called the adult T-cell lymphoma virus type 1. HTLV-II shares approximately 70% genetic homology (translating into 80-95% structural similarity at the protein level) with HTLV-I. The pathogenic potential of HTLV-II is not yet completely elucidated but HTLV-II is regarded as a risk marker for blood transfusion since it is mainly found in intravenous drug users world-wide (Vandamme et al., Evolutionary strategies of human T-cell lymphotropic virus type II, Gene 261 (2000) 171-180). Both viruses are spread globally, but the prevalence of HTLV-I is highest in hot spot regions in Southern Japan (Kyushu, Shikoku and Okinawa), Sub-Saharan Africa, the Caribbean (Jamaica and Haiti) and South America.
The major transmission modes of HTLV-I/II are through sexual contact, blood transfusion, sharing injection needles and mother to child transmission through breast-feeding. The seroconversion period after HTLV infection is long when compared to other infectious diseases. The window period, i.e. the time frame after infection within which no antibodies against the virus can be detected may range from several weeks to months.
Blood donor screening for HTLV was introduced first in Japan in 1986, in the United States and Canada in 1988/1989, in France in 1991 and in several European and South American countries after 1991. So far no gold standard has emerged for the diagnosis of HTLV infection. Several immunoassays based on recombinant and/or synthetic peptide antigens have been introduced in the past years.
Commercially available immunoassays for detecting anti-HTLV-antibodies often use polypeptides derived from the envelope of the virus (gp46 surface protein and gp21 transmembrane protein) or from the gag-encoded p24 capsid protein.
Due to the long seroconversion time it is important to detect even very small amounts of antibodies once they appear at an early stage after infection. Therefore, the development of appropriate antigens for a highly sensitive immunoassay is mandatory. As a matter of course, it is desirable to close the diagnostic gap between infection and detection, in order to prevent inadvertent spread and propagation of the virus.
It has been known for some time that, upon HTLV-infection, antibodies to the gag proteins appear early in seroconversion. In particular, the gag-encoded capsid antigen p24 is a preferred early target of the humoral immune response (Manns et al., Blood (1991) 77: 896-905). Hitherto, peptidic and recombinant variants of the p24 capsid protein have been used as antigens in immunoassays. By means of these antigens, anti-p24 immunoglobulins of the G-type have been detected with high accuracy and satisfying sensitivity. p24 capsid antigens of this kind are, however, not able to bind and detect immunoglobulins of the M type. Since IgM molecules usually appear before IgG molecules during seroconversion, we reasoned that it should be worthwhile to modify recombinant p24 capsid antigen in a way that it is recognized and bound by IgM. In brief, we wondered whether it was possible to improve the sensitivity of anti-p24 immunoglobulin detection by tailoring and engineering the p24 capsid antigen. In particular, we were seeking to design a p24 variant which was able to interact with and detect IgM molecules.
The problem underlying the invention therefore is the development of an immunoassay for detecting antibodies against HTLV-I and HTLV-II that overcomes the limited seroconversion sensitivity of the hitherto available immunoassays.
The problem is solved by the current invention as specified in the claims.